Journal:Analysis of phenolic compounds in commercial Cannabis sativa L. inflorescences using UHPLC-Q-Orbitrap HRMS

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Full article title Analysis of phenolic compounds in commercial Cannabis sativa L. inflorescences using UHPLC-Q-Orbitrap HRMS
Journal Molecules
Author(s) Izzo, Luana; Castaldo, Luigi; Narváez, Alfonso; Graziani, Giulia; Gaspari, Anna; Rodríguez-Carrasco, Yelko; Ritieni, Alberto
Author affiliation(s) University of Naples "Federico II," University of Valencia
Primary contact Email: luana dot izzo at unina dot it
Editors Efferth, Thomas
Year published 2020
Volume and issue 25(3)
Article # 631
DOI 10.3390/molecules25030631
ISSN 1420-3049
Distribution license Creative Commons Attribution 4.0 International
Website https://www.mdpi.com/1420-3049/25/3/631/htm
Download https://www.mdpi.com/1420-3049/25/3/631/pdf (PDF)

Abstract

Industrial hemp (Cannabis sativa L., family Cannabaceae) contains a vast number of relevant bioactive organic compounds, namely polyphenols, including flavonoids, phenolic acids, phenol amides, and lignanamides, which are well known for their therapeutic properties. Nowadays, many polyphenol-containing products made from herbal extracts are marketed, claiming to have health-promoting effects. In this context, industrial hemp inflorescences may represent an innovative source of bioactive compounds to be used in nutraceutical formulations.

The aim of this work was to provide a comprehensive analysis of the polyphenolic fraction contained in polar extracts of four different commercial cultivars—Kompolti, Tiborszallasi, Antal, and Selected Carmagnola (CS)—of hemp inflorescence through spectrophotometric (total phenolic content [TPC] and DPPH assays) and spectrometry measurement (using ultra high-performance liquid chromatographyquadrupoleorbitrap high-resolution mass spectrometry or UHPLC-Q-Orbitrap HRMS).

Results highlighted a high content of cannflavin A and B in inflorescence samples, which appear to be cannabis-specific, with a mean value of 61.8 and 84.5 mg/kg, meaning a ten-to-hundred times increase compared to other parts of the plant. Among flavonols, quercetin-3-glucoside reached up to 285.9 mg/kg in the CS cultivar. Catechin and epicatechin were the most representative flavan-3-ols, with a mean concentration of 53.3 and 66.2 mg/kg, respectively, for all cultivars. TPC in inflorescence samples was quantified in the range of 10.51 to 52.58 mg GAE/g, and free radical-scavenging included in the range from 27.5 to 77.6 mmol trolox/kg. As such, C. sativa inflorescence could be considered as a potential novel source of polyphenols intended for nutraceutical formulations.

Keywords: Cannabis sativa L., polyphenols, UHPLC-Q-Orbitrap HRMS

Introduction

Cannabis sativa is an annual herbaceous plant of the Cannabaceae family, native to Central Asia but with a wide distribution over different geographical areas, facilitated by climate adaptation. This plant has long been cultivated due to its large variety of applications, from textile uses to food and feed.[1]

Industrial hemp, characterized by a low content of psychoactive cannabinoids, contains bioactive organic compounds that are known to have a wide range of important biological properties.[2] Polyphenols represent one of the most relevant compounds found in C. sativa, including the likes of prenylated flavonoids, phenol amides, and lignanamides, which are specific metabolites of this plant. They are known to play multifunctional roles in the defense mechanisms of the plant, especially through their activity as antioxidants, preventing the generation of reactive oxygen species (ROS).[3][4][5][6] In humans, polyphenols can display health-promoting effects based on the modulation of several enzymes, such us lipoxygenase and the superfamily of cytochrome P450, showing cardio- or chemoprotective activity, among others.[5][7]

For this reason, polyphenol-containing products have been increasingly marketed as food supplements and nutraceuticals, and, currently, a great variety of supplements claiming to enhance specific physiological functions are commercially available. Nutraceuticals consist of naturally-occurring active substances, which are concentrated and administered in the suitable pharmaceutical form to properly develop its pharmacological effect. Furthermore, when compared to traditional drugs, nutraceuticals appear to be generally safer, with higher bioavailability and fewer side effects.[8] The manufacturing of nutraceuticals requires isolated ingredients that have to be extracted and purified for latter uses. Since certain polyphenols naturally occur inside insoluble structures, such as vacuoles, obtention of pure compounds can become a complex process.[9] In addition, several studies reported a decrease in the bioavailability and bioaccessibility of pure polyphenols in comparison with the administration of plant extracts rich in polyphenols, which may be due to the existence of other active compounds which can establish synergistic functions with them.[10][11][12] Because of this, food supplements could be a valuable resource to consume polyphenol-containing products. They consist of extracts from herbals and botanicals than can be delivered as the same pharmaceutical forms as nutraceuticals. Some of the most prevalent plants used as a source of polyphenols are tea, coffee, apple, basil, and turmeric, among others, each one intended for specific polyphenols.[13][14][15]

Regarding C. sativa, recent studies have reported the high antioxidant potential of the plant—while also characterizing the major polyphenols, N-trans-caffeoyltyramine, and cannabisin A, B, and C—and have concluded that C. sativa would be a suitable source of polyphenols for nutraceutical or supplementation purposes.[3][4][16][17][18] Nevertheless, the most studied components of the plant are seeds, leaves, and sprouts, whereas there is still scarce literature regarding polyphenols in inflorescences. The polyphenolic profile of C. sativa is variable among the different parts of the plant, and since flowers represent an important reproductive organ, high levels of colored polyphenols are expected.[19]

Analysis of polyphenols in C. sativa samples has been previously performed using Fourier transform infrared spectroscopy(FTIR) with attenuated total reflectance (ATR)[4], as well as mass spectrometry (MS) coupled to both high-performance liquid chromatography (HPLC) and gas chromatography (GC).[18] High-resolution mass spectrometers, such as Orbitrap, have also been used coupled to ultra high-performance liquid chromatography (UHPLC) for the determination of polyphenols in vegetal matrices intended for nutraceutical purposes, including green tea and coffee.[17][20][21][22][23] This methodology offers higher sensitivity and specificity, allowing a precise quantification based on exact mass measurement. Given this, this study aimed to (i) evaluate the antioxidant activity and total polyphenol content in different chemotypes of commercial C. sativa inflorescences using in vitro assays, and (ii) establish the polyphenolic profile of those inflorescences through UHPLC coupled to high-resolution Orbitrap mass spectrometry (UHPLC-Q-Orbitrap HRMS), leading to the promotion of this innovative source of bioactive compounds for use in nutraceutical formulations or for their health-promoting properties.

Results and discussion

Identification of polyphenol compounds in C. sativa inflorescences though UHPLC-Q-Orbitrap HRMS

Identification of individual phenolic acids and flavonoids was conducted through UHPLC-Q-Orbitrap HRMS. By a combination of MS and tandem mass spectrometry (MS/MS) spectra, a total of 22 different polyphenolic compounds were identified from different samples of C. sativa inflorescences (see Figures S1 and S2, Supplementary materials). Table 1 shows all mass parameters, including adduct ion, theoretical and measured mass (m/z), accuracy, and sensitivity.

Table 1. Chromatographic and spectrometric optimized parameters, including retention time, adduct ion, theoretical and measured mass (m/z), accuracy, and sensitivity for the investigated analytes (n = 22)
Compound Retention Time (min) Chemical Formula Adduct Ion Theoretical Mass (m/z) Measured Mass (m/z) Accuracy (Δ mg/kg) LOD (mg/kg) LOQ (mg/kg)
Catechin 7.65 C15H14O6 [M − H]− 289.07176 289.07224 1.6605 0.0015 0.0046
Chlorogenic acid 8.13 C16H18O9 [M − H]− 353.08780 353.08798 0.5098 0.0012 0.0036
Caffeic acid 8.24 C9H8O4 [M − H]− 179.03498 179.03455 −2.4018 0.0007 0.0020
Epicatechin 8.51 C15H14O6 [M − H]− 289.07176 289.07196 0.6919 0.0014 0.0043
Luteolin-7-O-glucoside 9.23 C21H20O11 [M − H]− 447.09328 447.09366 0.8499 0.0008 0.0025
p-Coumaric acid 9.31 C9H8O3 [M − H]− 163.04001 163.03937 −3.9254 0.0006 0.0018
Caffeoyl tyramine 9.46 C17H17NO4 [M − H]− 298.10848 298.10910 2.0798 - -
Rutin 9.79 C27H30O16 [M − H]− 609.14611 609.14624 0.2134 0.0012 0.0035
Ferulic acid 9.88 C10H10O4 [M − H]− 193.05063 193.05016 −2.4346 0.0018 0.0054
Quercetin-3-glucoside 9.93 C20H20O12 [M − H]− 463.08820 463.08862 0.9070 0.0017 0.0052
Kaempferol-3-O-glucoside 10.36 C21H20O11 [M − H]− 447.09323 447.09360 0.8276 0.0008 0.0025
Apigenin-7-glucoside 10.36 C21H20O10 [M − H]− 431.09837 431.09836 −0.0232 0.0004 0.0013
Cannabisin A 10.54 C34H30N2O8 [M − H]− 593.19294 593.19281 −0.2192 - -
Quercetin 11.00 C15H10O7 [M − H]− 301.03538 301.03508 −0.9966 0.0021 0.0064
Luteolin 11.25 C15H10O6 [M − H]− 285.04046 285.04050 0.1403 0.0004 0.0012
Cannabisin B 11.41 C34H32N2O8 [M − H]− 595.20859 595.20709 −2.5201 - -
Kaempferol 11.60 C15H10O6 [M − H]− 285.04046 285.04086 1.4033 0.0005 0.0014
Naringenin 11.78 C15H12O5 [M − H]− 271.06120 271.06146 0.9592 0.0005 0.0015
Apigenin 11.85 C15H10O5 [M − H]− 269.04555 269.04572 0.6319 0.0004 0.0011
Cannabisin C 12.34 C35H34N2O8 [M − H]− 609.22424 609.22485 1.0013 - -
Cannabisin B 13.77 C21H20O6 [M − H]− 367.11871 367.11871 0.0000 - -
Cannabisin A 14.84 C26H28O6 [M − H]− 435.18131 435.18143 0.2757 - -

Experiments were achieved in ESI− mode. All of the studied analytes exhibited better fragmentation patterns producing the quasi-molecular ion [M − H]−. After full scan analysis, the accurate mass of the characteristic ions (precursor ions) was included in an inclusion list.

Full-scan HRMS data acquisition captures all sample data, enabling the identification of untargeted compounds and retrospective data analysis without the need to re-run samples. The confirmation of the structural characterization of untargeted analytes was based on the accurate mass measurement, elemental composition assignment, and MS/MS spectrum interpretation (see Figure S3, Supplementary materials).

Optimal separation of all the investigated analytes was carried out in a total run time of 20 minutes. The identification of structural isomers—catechin and epicatechin (m/z 289.07176); luteolin and kaempferol (m/z 285.04046)—was achieved by comparing the retention times of the peaks with those of standards (see Figure S2, Supplementary materials).

Sensitivity was evaluated by the limit of detection (LOD) and limit of quantification (LOQ). The LOD was defined as the minimum concentration, where the molecular ion could be identified with a mass error below 5 ppm, and the LOQ was set as the lowest concentration of the analyte that produced a chromatographic peak with a precision and accuracy <20%.

Quantitative determination of target analytes (n = 16) was performed using calibration curves at eight concentration levels. Each calibration curve was prepared in triplicate. We obtained regression coefficients >0.990. Quantification of compounds (n = 6) that had no standard to generate a curve was based on a representative standard of the same group.

Quantification of phenolic acids and flavonoids in C. sativa inflorescences

Phenolic acids

The predominant lignanamides (cannabisin A, B, and C) and phenolic amide (N-trans-caffeoyltyramine) found in hemp were evaluated in the assayed samples. Lignanamides and phenolic amides belong to the lignan class of compounds, and the basic unit consists of tyramine condensed with CoA-esters of p-coumaric, caffeic, and coniferic acid, as suggested by Flores-Sanchez.[24] Table 2 shows the results here obtained, expressed as the average content and concentration range of the phenolic acids and flavonoids detected in different hemp varieties. In the here analyzed samples, lignanamides represented from 0.02% to 0.47% of total polyphenols, in a concentration range between 0.10 and 2.2 mg/kg. Cannabisin A was found as the most commonly detected lignanamide, ranging from 0.01 (Tiborszallasi) up to 2.86 mg/kg (Kompolti), with a mean value of 1.0 mg/kg for all cultivars. Cannabisin B was found at levels three times lower with respect to Cannabisin A, ranging from 0.4 to 0.5 mg/kg. In addition, when cannabisin A was found at very low concentrations, cannabisin B was not detected. Cannabisin C showed to be the least relevant lignanamide, quantified between 0.003 and 0.38 mg/kg. Concerning the occurrence of lignanamides in C. sativa seed, available studies reported the highest concentration up to thousands of milligram per kilogram.[25] As far as phenol amides were concerned, N-trans-caffeoyltyramine was quantified at a concentration range from 0.1 (Kompolti) to 76.2 mg/kg (CS), with a mean value of 23.7 mg/kg for all cultivars. These levels are in line with the data reported on hemp seed.[25]

Lignanamides and phenolic amides are known to have a wide range of important biological properties, including antioxidant, anti-inflammatory, and antihyperlipidemic activities.[26][27][28][29][30] Apart from those, some important hydroxycinnamic acids (chlorogenic acid, caffeic acid, p-coumaric acid, and ferulic acid) were also evaluated in the here analyzed inflorescences samples. This important class of phenolic acids represented from 18.6% to 29.7% of total polyphenols found in samples. Among the hydroxycinnamic acids, p-coumaric acid was quantified at concentrations significantly greater (p < 0.05) than the other related compounds in all hemp cultivars analyzed, except in Kompolti samples. Moreover, the most common hydroxycinnamic acids found in Kompolti cultivar was ferulic acid, at an average content of 19.7 mg/kg (range from 3.0 to 35.6 mg/kg). Caffeic acid was detected in the lowest amount for all the analyzed cultivars. The CS hemp variety showed the highest concentration of hydroxycinnamic acids, compared with other varieties at an average content of 85.4 mg/kg. On the other hand, the observed concentration variability of phenolic acids may be a result of the influence of many biotic and abiotic factors that play an important role in the biosynthetic process of the studied compounds.[31]

Table 2. Polyphenol content in the analyzed Cannabis sativa samples (n = 22). Results are shown based on the different cultivar C. sativa inflorescences.
Sample Kompolti (n = 9) Tiborszallasi (n = 7) Antal (n = 7) Selected Carmagnola (CS) (n = 4)
Average (mg/kg) Range (mg/kg) Average (mg/kg) Range (mg/kg) Average (mg/kg) Range (mg/kg) Average (mg/kg) Range (mg/kg)
Phenolic acids
Hydroxycinnamic acids
Chlorogenic acid 12.0 2.2–28.2 9.0 2.0–20.5 10.1 3.5–23.6 15.0 11.1–22.1
Caffeic acid 1.4 0.4–2.8 4.3 1.2–6.4 3.3 1.6–5.6 3.9 2.9–4.6
p-Coumaric acid 13.1 0.5–28.0 37.3 15.5–84.7 28.2 5.1–105.8 41.1 18.1–93.0
Ferulic acid 19.7 3.0–35.6 26.0 14.7–35.3 18.9 4.7–30.6 25.5 20.2–33.4
   Totals 46.2 76.5 60.5 85.4
Lignanamides
Cannabisin A 1.1 0.1–2.9 0.01 0.005–0.01 1.5 1.2–1.8 1.6 0.09–2.85
Cannabisin B 0.40 0.02–1.1 - - 0.6 0.4–0.7 0.5 0.02–1.15
Cannabisin C 0.10 0.01–0.35 0.09 0.01–0.27 0.14 0.01–0.38 0.02 0.003–0.05
   Totals 1.60 0.1 1.7 2.12
Phenolic amides
N-trans-Caffeoyltyramine 17.6 0.1–59.2 15.3 4.7–30.6 25.8 5.7–44.9 36.1 5.3–76.2
   Totals 17.6 15.3 25.8 36.1
Flavonoids
Flavonols
Rutin 13.8 1.7–42.8 27.6 14.1–38.6 12.5 2.4–24.1 27.2 7.8–60.8
Quercetin-3-glucoside 37.6 2.2–87.1 94.3 23.2–269.8 55.8 10.0–172.1 126.1 58.9–285.9
Kaempferol-3-O-glucoside 6.5 0.1–15.3 15.4 4.2–44.5 9.9 1.6–29.6 26.6 12.6–46.6
Quercetin 12.1 6.2–24.6 15.8 6.3–26.0 10.3 6.5–16.8 28.8 8.2–58.5
Kaempferol 3.4 0.7–7.6 5.1 1.3–8.7 2.4 0.3–3.9 9.2 0.6–13.6
   Totals 73.4 158.2 90.9 217.8
Flavones
Cannflavin A 55.4 35.2–130.0 72.9 30.9–107.5 51.3 19.6–106.5 67.7 28.4–118.6
Cannflavin B 55.4 30.2–110.6 86.7 26.2–215.5 98.8 32.9–182.5 97.2 11.9–154.4
Luteolin-7-O-glucoside 9.4 3.3–19.0 13.4 2.1–42.8 13.9 8.8–17.7 22.2 1.1–52.6
Apigenin-7-O-glucoside 1.9 0.1–5.6 2.5 0.1–6.7 1.1 0.2–3.0 2.6 0.8–6.5
Luteolin 12.2 0.8–23.5 19.0 6.3–38.2 8.6 0.9–14.3 25.7 16.9–38.2
Apigenin 5.4 2.1–11.7 6.5 2.4–16.3 4.1 0.4–8.5 8.7 6.3–13.9
   Totals 139.7 201.0 27.7 59.2
Flavan-3-ols
Catechin 85.3 0.1–334.0 16.8 2.6–43.4 40.9 1.9–155.2 70.1 39.2–115.2
Epicatechin 30.7 1.6–88.4 64.2 10.6–183.7 63.2 11.1–156.4 11.1–156.4 21.5–194.6
   Totals 116.0 81.0 104.1 177.0
Flavanone
Naringenin 0.50 0.01–1.10 0.5 0.3–1.0 0.9 0.5–2.0 1.0 0.7–1.8
   Totals 0.50 0.5 0.9 1.0
   Total bioactive 377.9 532.6 461.7 743.5

Flavonoids

Flavonoids are plant-derived phytochemicals that accounted for over 80% of the phenolic components in the assayed samples. Flavonoids—namely flavones, flavanones, flavonols, and flavan-3-ols, including their main aglycones, glycosides, and methylated derivatives—have been quantified in the assayed samples, as shown in Table 2, above. Flavones represented the highest proportion of flavonoids found in analyzed samples, ranging from 30.1% (CS) to 35.4% (Kompolti) of total polyphenols, cannflavin A and cannflavin B being the most commonly detected flavones, with a mean value of 61.8 and 84.5 mg/kg, respectively. These levels found in inflorescences showed a ten-to-hundred-fold increase when compared to leaf-based samples previously analyzed by Pollastro et al.[5], who reported cannflavin A and B at 6 and 0.8 mg/kg, respectively. Cannflavins A and B, methylated isoprenoid flavones, appear to be Cannabis-specific and are known to exert a potent anti-inflammatory activity.[32] In this context, Werz et al.[33] reported that cannflavin A and B were able to inhibit the production of pro-inflammatory prostanoids and leukotrienes in in vitro assays. Moreover, Barrett et al. concluded that cannflavins A and B promoted the inhibition of PGE2 in human cells up to thirty times in relation to aspirin.[34][35] In addition, cannflavin A showed a neuroprotective effect against amyloid β-mediated neurotoxicity in PC12 cells.[36] As shown in Table 2, among flavonols, quercetin-3-glucoside was quantified at concentrations significantly greater (p < 0.05) than the other related compounds, ranging from 2.2 (Kompolti) to 285.9 mg/kg (CS), with a mean value of 78.4 mg/kg. On the other hand, flavan-3-ols, mainly represented by catechin and epicatechin, were detected at mean values of 53.3 and 66.2 mg/kg for all cultivars, respectively. Regarding epicatechin, the CS cultivar showed a two-fold increase compared with other cultivars assayed. Of the minor flavonoids, the compound naringenin was detected.

Overall, the data clearly indicate that the CS cultivar showed the highest concentrations of the investigated polyphenols (743.5 mg/kg) compared to the other cultivars.

Industrial hemp inflorescence may represent an innovative source of bioactive compounds—such as a high content of cannflavin A and B, which appear to be Cannabis-specific—to be used in nutraceutical formulations.

Total phenolic contents and antioxidant activity of C. sativa extracts

Total phenolic contents of C. sativa extracts

The amounts of total phenolic contents of C. sativa extracts were examined. The results are summarized in Table 3. Total phenolics, flavonoids, and phenolic acids ranged from 10.510 to 48.875 mg GAE/g for the different cultivars of C. sativa. Among the studied cultivars, the highest total phenol content resulted from the CS cultivar, with a content of 41.517 mg GAE/g, and the lowest amount emerges for the Kompolti variety (10.510 mg GAE/g). Total polyphenol content quantified in analyzed samples was similar to those recently reported by Vonapartis et al.[37], who found hemp seed samples (n = 10) to be in a concentration range from 13.68 to 51.60 mg GAE/g.

Table 3. Total polyphenol compounds expressed as mg GAE/g and antioxidant activity expressed as mmol trolox/kg of C. sativa extracts; GAE: gallic acid equivalents, TPC: total phenolic content, DPPH: 2,2-diphenyl-1-picrylhydrazyl free-radical scavenging, trolox: equivalent antioxidant capacity (TEAC)
Cultivar TPC DPPH
Average (mg GAE/g) Range (mg GAE/g) Average (mmol trolox/kg) Range (mmol trolox/kg)
Kompolti (n = 9) 26.2 ± 0.5 10.5–47.2 46.7 ± 0.7 36.6–55.0
Tiborszallasi (n = 7) 29.9 ± 0.4 21.9–42.9 61.3 ± 0.9 50.8–72.7
Antal (n = 7) 30.7 ± 0.5 17.0–48.9 45.9 ± 0.4 27.5–67.5
Selected Carmagnola (CS) (n=4) 33.2 ± 0.5 26.3–41.5 63.6 ± 0.9 59.1–77.6

C. sativa is a remarkable plant widely investigated by several researchers, given its rich fount of valuable natural components. Apart from cannabinoid production, the C. sativa plant is also able to synthesize non-cannabinoid secondary metabolites possessing beneficial effects for human health.[1] [38] A summary of the available surveys on total polyphenol content of the different parts of C. sativa is shown in Table 4. In spite of several experiments being performed on this plant[3][4][17][38][39][40][41][42][43], the study of inflorescences remains low, making the comparison of results difficult. Recently, Ferrante et al.[42] investigated the total phenolic content in the water fraction extracted from aerial flowers of the CS cultivar, reporting a lower value compared to the results here obtained.


Supplementary materials

Supplementary file 1: This PDF file contains Supplementary Figures S1–S3.

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Notes

This presentation is faithful to the original, with only a few minor changes to presentation. Some grammar and punctuation was cleaned up to improve readability. In some cases important information was missing from the references, and that information was added.